Encapsulated microorganisms and methods of using same

ABSTRACT

A dehydrated composition containing a particle encapsulating one or more microorganisms is provided. The composition is useful for controlling the release of the microorganisms following rehydration and propagation within the particle.

FIELD OF THE INVENTION

The present invention relates to particles capable of releasing viablemicroorganisms encapsulated therein following a preset incubation andpropagation period.

BACKGROUND OF THE INVENTION

Microorganisms are increasingly being used in agricultural,environmental and medical applications. In agriculture, microorganismscan be applied to plants or soil in order to increase crop productivity,protect plants from diseases and maintain soil viability.

When utilized in open settings (e.g., agriculture, environmentalprotection), microbial cells can be diluted, dispersed andinactivated/killed by the environment before providing the desiredeffect. To overcome this problem, higher starter doses of microbialcells are utilized requiring culturing to excess in advance of use.

Approaches for packaging of microbial cells for transport/delivery havebeen described and typically involve desiccation of microbial cells inorder to maintain stability and sterility of the microbial product untilactivation. Dried microbial cells can be rehydrated at the targetenvironment to reestablish the desired activity. However, drying canresult in death of most of the packaged microbial cells.

U.S. Pat. No. 7,422,737 discloses cellular solid carriers comprisingviable microorganisms capable of controlling plant pathogens. Thecellular solid carriers are formed from water-soluble hydrocolloid beadsdried under conditions which preserve their porosity, thereby allowingefficient release of microorganisms or diffusion of products derivedfrom the microorganisms from the beads to the surrounding environment.

Przyklenk et al. (Journal of Microencapsulation, DOI:10.1080/02652048.2017.1354941 (2017)) discloses the development ofencapsulated and dried entomopathogenic fungus Metarhiuzm brunneum withreduced conidia content, increased conidiation, a high drying survivaland enhanced shelf life. Dried beads were prepared with corn starch,potato starch, carboxymethylcellulose or autoclaved baker's yeast asfillers.

There is thus a need for, and it would be highly advantageous to have, acomposition that encapsulates microorganisms and enables theirpropagation and timely release at a target environment such as a cropfield or greenhouse.

SUMMARY OF THE INVENTION

The present invention is conducive to the propagation and release ofmicroorganisms at a target environment in a manner that enhances theirability to survive in that environment. The present invention thusprovides a composition that includes encapsulated microorganism(s) whichcan be propagated and released at a target environment in a controlledmanner. The composition comprises a particle (e.g. a microparticle)encapsulating one or more viable microorganisms, wherein the particlecomprises an inner core (lumen) containing the one or moremicroorganisms, optionally together with the nutrients required fortheir propagation, the inner core being surrounded by an outer shelllayer which delays the dispersal of microorganisms from within theparticle to the environment. The particle is dried and optionally storedbefore its application to the environment of choice. The outer shelllayer is permeable to water but does not enable the release ofmicroorganisms encapsulated therein for a predetermined period of timeafter rehydration. Hence, the microorganisms are maintained or retainedwithin the particles until they have multiplied and can be releasedsuccessfully to the environment in which they are required with greaterchances of survival. The composition of the present invention thusprovides the release of viable microorganisms in culture density or cellcount suitable for uses such as increasing crop productivity, protectingplants from diseases and maintaining soil viability, even when theculture density or cell count prior to rehydration is low.

According to one aspect of the present invention, there is provided adehydrated composition comprising a particle encapsulating one or moremicroorganisms, wherein the particle is composed of an inner corecomprising the one or more microorganisms surrounded by an outer shelllayer, wherein said outer shell layer is selectively permeable to arehydrating fluid, and wherein upon fluid absorption, said outer shelllayer degrades at a predetermined rate thereby releasing a plurality ofmicroorganisms to the surrounding environment in a controlled manner,wherein the encapsulated microorganisms are present in the dehydratedcomposition at an initial concentration of less than about 1×10⁵ colonyforming units (CFU), and wherein following fluid absorption, theconcentration of the encapsulated microorganisms is increased by atleast 10-fold before the microorganisms are released to the surroundingenvironment. In certain embodiments, the dehydrated compositioncomprises a plurality of particles.

In various embodiments, the outer shell layer is designed to degradegradually upon fluid absorption. In other embodiments, the outer shelllayer is designed to be digested by the microorganisms containedtherein. In further embodiments, the outer shell layer is sensitive topH. In yet further embodiments, the outer shell layer islight-sensitive. In additional embodiments, the outer shell layer issensitive to temperature changes. In other embodiments, the outer shelllayer is sensitive to changes initiated by the propagation of themicroorganisms thereby releasing the microorganisms in a controlledmanner.

In some embodiments, the outer shell layer comprises a naturallyoccurring polymer, a synthetic polymer, or a semi-synthetic polymer,with each possibility representing a separate embodiment. In variousembodiments, the outer shell layer comprises a water-degradable polymer.In certain embodiments, the polymer is selected from the groupconsisting of polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP),polyethylene oxide (PEO), polyethylene glycol (PEG), polycaprolacetone(PCL), polyurethane (PU), polyglycolic acid (PGA), polylactic acid(PLA), polylactic-co-glycolic acid (PLGA), poly-L-lactic acid (PLLA),cellulose derivatives, chitosan, chitin, hyaluronan (HA), gelatin, nylon6, polyacrylonitrile (PAN), polylactide/polyhydroxybutyrate (PLA/PHB),alginate polymer, and a mixture or combination thereof. Each possibilityrepresents a separate embodiment. In one embodiment, the polymer is PVA.In another embodiment, the polymer is gelatin. In yet anotherembodiment, the polymer is an alginate polymer.

In certain embodiments, the outer shell layer is formed from a polymeror polymer precursor configured to undergo a phase transition inresponse to a stimulus. In certain embodiments, the stimulus ischemically-induced. In other embodiments, the stimulus isphysically-induced. In particular embodiments, the stimulus comprises achange in at least one of temperature, pH, light, and electric field,with each possibility representing a separate embodiment.

According to various embodiments, the composition is dehydrated to awater content of about 0.5% to about 10% by weight of the total weightof the composition, including each value within the specified range. Itis contemplated that the outer shell layer of the particle isselectively permeable to a rehydrating fluid whereby upon rehydration(i.e. fluid absorption), propagation of the microorganism(s) within theparticle is afforded followed by partial or complete degradation of theouter shell layer of the particle to provide the controlled release ofthe microorganisms encapsulated therein.

According to embodiments of the present invention, the particle has anaverage size ranging from about 1 to about 9,999 microns in diameter,including each value within the specified range. In some embodiments,the particle has an average size ranging from about 1 to about 100microns in diameter, including each value within the specified range. Incertain embodiments, the particle is at least 50 microns in diameter,for example about 50 to about 100 microns in diameter, including eachvalue within the specified range. In additional embodiments, the averagesize of the particle is less than 100 microns in diameter.

According to other embodiments of the present invention, the particleencapsulates microorganisms at an initial concentration of less thanabout 1×10⁵ colony forming units (CFU), for example about 1 to about1×10⁵ CFU, including each value within the specified range. In furtherembodiments, the particle encapsulates microorganisms at an initialconcentration of less than about 1×10⁴ CFU, for example about 1 to about1×10⁴ CFU, including each value within the specified range. In exemplaryembodiments, the particle encapsulates microorganisms at an initialconcentration of less than about 1×10³ CFU, for example about 1 to about1×10³ CFU, including each value within the specified range. In yet otherembodiments, the particle encapsulates microorganisms at an initialconcentration of about 10 to about 1,000 CFU, including each valuewithin the specified range.

It is contemplated that, upon rehydration, microincubation andpropagation of the microorganism occurs, thereby resulting in asignificant increase in the number of encapsulated microorganisms. Inone embodiment, upon rehydration (fluid absorption), the concentrationof the encapsulated microorganisms is increased by at least 2, 3, 4, 5,6, 7, 8, 9, or even 10-fold as compared to naked non-encapsulatedmicroorganisms. In certain embodiments, upon rehydration, theconcentration of the encapsulated microorganisms is increased by atleast 10-fold before the microorganisms are released to the surroundingenvironment. In another embodiment, upon rehydration, the concentrationof the encapsulated microorganisms is increased by at least 20-foldbefore the microorganisms are released to the surrounding environment.In yet other embodiments, upon rehydration, the concentration of theencapsulated microorganisms is increased by at least 30-fold before themicroorganisms are released to the surrounding environment. In furtherembodiments, the concentration of the encapsulated microorganisms isincreased by at least 10-fold 24 hours following fluid absorption.

According to embodiments of the present invention, the microorganismsare released from the particle after a preset period. In one embodiment,a partial or complete degradation of the outer shell layer of theparticle is designed to afford the release of the microorganismsinitiating from about 24 hours to about 7 days following rehydration,including each value within the specified range. In certain embodiments,the outer shell layer of the particle is designed to have a degradationrate that affords the release of the microorganisms initiating fromabout 24 to about 96 hours following rehydration, including each valuewithin the specified range. In other embodiments, the outer shell layerof the particle is designed to have a degradation rate that affords therelease of the microorganisms initiating from about 24 to about 72 hoursfollowing rehydration, including each value within the specified range.In further embodiments, the release of the microorganisms is immediateafter a preset period, i.e. a delayed release. In additionalembodiments, the release of the microorganisms is modified by thepresence of a sustained release agent within the inner core, therebyproviding prolonged release of the microorganisms after a predeterminedlag time. In accordance with these embodiments, the sustained releaseagent comprises a polymer which may be the same or different from thepolymer forming the outer shell layer, with each possibilityrepresenting a separate embodiment.

According to further embodiments of the present invention, the innercore may further comprise one or more nutrients to support the growth ofthe encapsulated microorganism. In some embodiments, the nutrientsinclude a carbon source, a nitrogen source, a phosphorous source, or amixture or combination thereof, with each possibility representing aseparate embodiment.

According to certain embodiments of the present invention, the outershell layer may further be coated with at least one other coating layer.It is contemplated that the outer shell layer may further be coated witha plurality of layers, each having a different functionality. Forexample, the coating layer may exert different permeability than theouter shell layer thereby further modifying the release of theencapsulated microorganisms. In another example, the coating layer maybe an adhesive coating layer thereby enabling the adhesion of theparticle to a plant tissue (e.g. a plant seed).

According to another aspect of the present invention, there is provideda plant tissue coated with or in the vicinity of the particle describedherein. Each possibility represents a separate embodiment. In oneembodiment, the plant tissue is a plant seed.

According to yet another aspect of the present invention, there isprovided a method of preparing a dehydrated composition comprising aparticle as described herein, the method comprising: suspending one ormore microorganisms in a solution optionally comprising one or morenutrients; encapsulating the one or more microorganisms and optionalnutrients in a polymer thereby obtaining a particle composed of an innercore comprising said one or more microorganisms and optional nutrientssurrounded by an outer shell layer; and dehydrating the particle. Insome embodiments, the step of encapsulating the one or moremicroorganisms and optional nutrients comprises adding a polymer orpolymer precursor to the suspension comprising the one or moremicroorganisms and optional nutrients, and inducing phase transition tothe polymer or polymer precursor thereby obtaining a particle composedof an inner core comprising said one or more microorganisms and optionalnutrients surrounded by an outer shell layer. In various embodiments,the phase transition comprises polymerization of the polymer precursor.In other embodiments, the phase transition comprises cross-linking ofthe polymer. In additional embodiments, the phase transition is effectedin response to a stimulus that may be chemically induced or physicallyinduced, with each possibility representing a separate embodiment. Inparticular embodiments, the stimulus comprises a change in at least oneof temperature, pH, light, and electric field, with each possibilityrepresenting a separate embodiment.

According to embodiments of the present invention, there is provided amethod of delivering a plurality of microorganisms to a targetenvironment comprising rehydrating a dehydrated composition comprising aparticle as described herein with a suitable amount of fluid therebyallowing the propagation and subsequent release of a plurality ofmicroorganisms from the particle in a controlled manner.

According to particular embodiments of the present invention, the fluidis water.

Target environments include, but are not limited to, soil, phyllosphere,rhizosphere, sewage reclamation, toxic spill, fermentation, and thegastrointestinal tract. Each possibility represents a separateembodiment. In some embodiments of the present invention, the targetenvironment is a soil environment.

In certain embodiments of the present invention, the microorganismscomprise endophytic bacteria.

Further embodiments and the full scope of applicability of the presentinvention will become apparent from the detailed description givenhereinafter. However, it should be understood that the detaileddescription and specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only,since various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thisdetailed description. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 schematically illustrates one or more cells of a microorganismand nutrients (supplemental materials) disposed within a dehydratedmicroparticle and the microincubation and controlled release of theencapsulated microorganisms following rehydration.

FIG. 2 schematically illustrates the steps of preparing one embodimentof the composition of the present invention.

FIGS. 3A-3B show graphs of comparative results of propagation in soil ofnaked or encapsulated Pseudomonas chlororaphis in different polymers atinitial concentrations of 10²-10⁵ CFU per 2 grams of soil (FIG. 3A) and10⁵-10⁶ CFU per 2 grams of soil (FIG. 3B) over 96 hours.

FIG. 4 shows a graph of comparative results of propagation in soil ofPseudomonas chlororaphis encapsulated in 10% PVA 99 h Mw=31K-50K and in5% CMC over 144 hours.

FIGS. 5A-5C illustrate the growth of Pseudomonas chlororaphis within acapsule, at time 0 (FIG. 5A), after 24 hours (FIG. 5B), and after 48hours (FIG. 5C).

FIG. 6 shows a graph of comparative results of bacterial release fromcapsules in an accelerated model.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a composition that includes encapsulatedmicroorganism(s) which can be propagated and released at a targetenvironment in a controlled manner.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Approaches for preparing and storing microorganisms are well known inthe art. While such approaches may be adequate for maintainingmicroorganisms viable until use, they are not suitable when themicroorganisms are used in open settings (e.g., agriculture,environmental protection, etc.) since such conditions can dilute,disperse and inactivate the microorganisms before providing the desiredeffect. Accordingly, open settings typically require the application ofvery high concentrations of microorganisms such that following theirdilution, dispersion and inactivation, there remains a sufficient amountof microorganisms that can exert their beneficial effects. However, itis not commercially feasible to use very high concentrations ofmicroorganisms. The present invention provides a surprisinglyadvantageous composition that enables prolongation of shelf-life andlowering the viability threshold of microorganisms, thus beingparticularly suitable for use in open settings.

According to certain aspects and embodiments, there is provided acomposition that can:

(i) maintain agriculturally/environmentally important microorganismsviable for extended period of time (months to years);

(ii) enable dispersal and release of a plurality of microorganisms in anenvironment such as a field in a controlled manner; and

(iii) time the release of the plurality of microorganisms to theenvironment following a period in which the microorganisms propagate toa predetermined culture density while not being exposed to theenvironment.

The present invention affords the propagation of microorganisms withinthe confinement of protecting capsules until they reach a high enoughconcentration that enables their successful release to a targetenvironment. This enables a reduction in the minimum initial dose ofmicroorganisms to less than 1×10⁵ colony forming units (CFU).

As is described hereinunder and in the Examples section which follows,there is provided a composition that includes one or more microorganismcells encapsulated within a particle. The particle is characterized byan inner core (lumen) surrounded by an outer shell layer which enablesthe selective inflow of a fluid into the particle to result in thepropagation of the microorganism(s) within the particle lumen whilepreventing outflow of microorganisms until propagation results in adesired culture density or cell count. The particle therefore performsas a microincubator to microorganisms which can be applied to plants orsoil for various uses including, but not limited to, increasing cropproductivity, protecting plants from diseases, and maintaining soilviability.

As used herein and in the appended claims, the term “particle” refers toany sub-centimeter scale particle (e.g. about 1 to about 9,999 micronsin size as measured along a selected axis, e.g. diameter). Typicaldimensions for a particle can be up to 10,000 microns in outer diameter(OD) and up to 9,990 microns in lumen diameter (LD). In someembodiments, the particle has an average size ranging from about 1 toabout 100 microns in diameter, including each value within the specifiedrange, for example about 1, about 5, about 10, about 15, about 20, about25, about 30, about 35, about 40, about 45, about 50, about 55, about60, about 65, about 70, about 75, about 80, about 85, about 90, about95, or about 100 microns. Each possibility represents a separateembodiment. In certain embodiments, the particle is at least 50 micronsin diameter. In other embodiments, the particle is less than 100 micronsin diameter. In further embodiments, the particle has an average size ofabout 50 to about 100 microns in diameter, including each value withinthe specified range. According to the principles provided herein, theparticle may have any shape suitable for encapsulation of amicroorganism within a lumen thereof. Exemplary shapes include, but arenot limited to, spherical, oval, cylindrical, cube, and the like. Eachpossibility represents a separate embodiment. More complex shapes suchas star, filament or sheet are also contemplated herein and may beadvantageous for use in certain environments as is further detailedhereinunder.

Examples of microorganisms that can be encapsulated and cultured withinthe particle include, but are not limited to, bacteria, archaea, fungi(including yeast), protists and other prokaryotic and eukaryoticmicroorganisms. Each possibility represents a separate embodiment. Oneor more types of prokaryotic and/or eukaryotic microorganisms or species(e.g. 1, 2, 3 or more species of bacteria) can be encapsulated within asingle particle. It is to be understood that the method and particle canbe applicable to any microorganism. Non-limiting examples ofmicroorganisms include:

Bacteria (Phylum Actinobacteria)—Arthrobacter agilis, Arthrobacteraurescens, Arthrobacter globiformis, Arthrobacter nitroguajacolicus,Curtobacterium flaccumfaciens, Curtobacterium sp., Kocuria palustris,Microbacterium arborescens, Microbacterium oxydans, Paenarthrobacternicotinovorans, Streptomyces lydius, and Streptomyces fulvissimus. Eachpossibility represents a separate embodiment.

Bacteria (Phylum Bacteroidetes)—Chryseobacterium lathyri, Flavobacteriumendphyticum, and Flavobacterium johnsoniae. Each possibility representsa separate embodiment.

Bacteria (Phylum Firmicutes)—Bacillus amyloliquefaciens, Bacillusaquimaris, Bacillus endophyticus, Bacillus lehensis, Bacillusmegaterium, Bacillus simplex, Paenibacillus sp., Bacillus subtilis,Bacillus toyonensis, Enterococcus casseliflavus, and Enterococcusfaecalis. Each possibility represents a separate embodiment.

Bacteria (Order Alphaproteobacteria)—Rhizobium alamii, Rhizobium sp.,Rhizobium leguminosarum, Rhizobium radiobacter, Rhizobium tropici,Shinella sp., Sinorhizobium meliloti, Sphingobium herbicidovorans,Ochrobactrum anthropi, Sphingobium yanoikuyae, Sphingomonashankookensis, Sphingomonas koreensis, Sphingomonas pseudosanguinis, andSphingomonas sanguinis. Each possibility represents a separateembodiment.

Bacteria (Order Betaproteobacteria)—Acinetobacter calcoaceticus,Acinetobacter sp., Enterobacter cloacae, Erwinia billingiae, Erwiniagerundensis, Lysobacter capsica, and Pantoea vagans. Each possibilityrepresents a separate embodiment.

Bacteria (Order Gammaproteobacteria)—Acinetobacter calcoaceticus,Acinetobacter sp., Enterobacter cloacae, Erwinia billingiae, Erwiniagerundensis, Lysobacter capsica, Pantoea vagans, Pseudacidovoraxintermedius, Pseudomonas alcaligenes, Pseudomonas benzenivorans,Pseudomonas borborid, Pseudomonas chlororaphis, Pseudomonasextremaustralis, Pseudomonas fluorescens, Pseudomonasfrederiksbergensis, Pseudomonas monteilii, Pseudomonas moraviensis,Pseudomonas plecoglossicida, Pseudomonas putida, Pseudomonasrizosphaerae, Pseudomonas sihuiensis, Pseudomonas stutzeri, Pseudomonastaiwanensis, Pseudomonas aeruginosa, Pseudoxanthomonas sacheonensis,Rosenbergiella sp., Serratia marcescens, Serratia nematodiphilia,Serratia plymuthica, Stenotrophomonas geniculate, Stenotrophomonasmaltophilia, Stenotrophomonas pavanii, and Stenotrophomonasnitritireducens. Each possibility represents a separate embodiment.

Archaea—Pyrococcus furiosus, Metallosphera sedula, Thermococcuslitoralis, Methanococcus jannaschii, Sulfolobus solfataricus,Methanobacterium thermoautotrophicum, and Aeropyrum pernix. Eachpossibility represents a separate embodiment.

Fungi (including yeast)—Trichoderma harzianum, Rhizophagus irregularis,Aspergillus awamori, Metarhizium anisopliae, Sarocladium spinificis,Saccharomyces cerevisiae, Debaryomyces hansenii, Yarrowia lipolytica,Kluyveromyces marxianus, Zygosaccharomyces rouxii, Pichia pastoris,Sarocladium implicatum, and Candida versatilis. Each possibilityrepresents a separate embodiment.

Protista—Protozoa, Protophyta (algae) and molds. Each possibilityrepresents a separate embodiment.

Other eukaryotes—various cell lines of various organisms (insect,mammalia) including stem cells such as embryonic stem cells, and primarycells such as epithelial cells. Each possibility represents a separateembodiment.

Currently preferred microorganisms include endophytic bacteria,particularly of species that are useful for agricultural use.

According to certain embodiments, the composition is dehydrated. As usedherein, the term “dehydrated” refers to a composition having a watercontent of about 0.5 to about 10% by weight of the total weight of thecomposition, including each value within the specified range. Forexample, the water content of the dehydrated composition may be about0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%,about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, or about 10%by weight of the total weight of the composition. Each possibilityrepresents a separate embodiment. Dehydration can be effected usingconventional methods such as lyophilization, oven drying, and the like.

According to some embodiments, the dehydrated composition comprises aparticle as described herein. According to other embodiments, thedehydrated composition comprises a plurality of particles as describedherein. The particle is configured to enable the release of a pluralityof microorganisms following rehydration of the composition andpropagation of the microorganism(s) within the particle. This enablesthe microorganism(s) to propagate within the particle to a density/cellcount which ensures viability and function in the target environment towhich it is released.

In order to support propagation of microorganisms, the particle canoptionally include growth nutrient(s) that provide a source of carbon,nitrogen, phosphorous (phosphates), potassium, and microelements to themicroorganisms (when in a fluid medium). The type and concentration ofsuch nutrients (in the rehydrating fluid within the microparticle) canbe determined by a person of ordinary skill in the art depending on thetype of microorganism encapsulated. Within the scope of the presentinvention is a dehydrated composition comprising a plurality ofparticles, each particle encapsulates a different species ofmicroorganism together with nutrients suitable for its propagation, theplurality of particles further surrounded by an outer shell layer asdescribed herein for the controlled release of the differentmicroorganisms from their respective particles in a controlled manner.

Examples of nutrients suitable for supporting propagation ofmicroorganisms are provided in the Examples section which follows.Typically, the nutrients include carbohydrates (e.g. glucose, sucrose,molasses, cellulose, and starch), vitamins, and the like. Furthermore,suitable growth media such as, but not limited to, Nutrient Broth (NB),Luria Bertini (LB) broth, Bushnell-Haas Broth (BHB), Trypticase SoyBroth (TSB), and the like may be used. Each possibility represents aseparate embodiment. In certain embodiments, the outer shell layer maybe biodegradable such that it provides the nutritional support to theencapsulated microorganisms. A degradable particle can also provide someor all of the nutrients needed to support microorganism growth. Forexample, alginate, cellulose derivatives, carrageenan, gelatin, andpectin particles are made of proteins or polysaccharides that can bedegraded to support the growth of the encapsulated microorganisms. Inaccordance with these embodiments, the outer shell layer partially orcompletely degrades due to digestion or consumption by the encapsulatedmicroorganisms.

According to certain aspects and embodiments of the present invention,the dehydrated composition provides the encapsulation of one or moremicroorganism cells at an initial concentration of less than about 1×10⁵colony forming units (CFU). The term “initial concentration” as usedherein refers to the concentration of viable microorganisms (single ormultiple types or species) just before rehydration, i.e. at time zerobefore fluid absorption prior to incubation and propagation. It iscontemplated that a concentration of microorganisms in freshly madecapsules prior to storage may be higher than 1×10⁵ CFU. However, duringstorage, the concentration of viable microorganisms is typically reducedsuch that the initial concentration at time zero before fluid absorptionis less than about 1×10⁵ CFU. According to the principles providedherein, due to loss of viability of cells during formulation andstorage, the concentration of microorganisms encapsulated within theparticle can be significantly lower than that required to be effectivein a target environment. A relatively small number of surviving cellscan grow back to occupy the lumen (interior) of the capsule using theprovided nutrients and nutrients derived from the dead cells. Even whenthe cell count prior to rehydration is low, the particle of the presentinvention is designed to perform as a microincubator upon rehydration toafford the release of a plurality of microorganisms to the targetenvironment only when the culture density or cell count reaches acertain predetermined value suitable for exerting the desired effect.

In some embodiments, the initial concentration of encapsulatedmicroorganisms is about 1 to about 1×10⁵ CFU, including each valuewithin the specified range, for example about 1, about 10, about 20,about 30, about 40, about 50, about 60, about 70, about 80, about 90,about 100, about 150, about 200, about 250, about 300, about 350, about400, about 450, about 500, about 550, about 600, about 650, about 700,about 750, about 800, about 850, about 900, about 950, about 1,000,about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about4,000, about 4,500, about 5,000, about 5,500, about 6,000, about 6,500,about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about9,500, about 10,000, about 15,000, about 20,000, about 25,000, about30,000, about 35,000, about 40,000, about 45,000, about 50,000, about55,000, about 60,000, about 65,000, about 70,000, about 75,000, about80,000, about 85,000, about 90,000, about 95,000, or about 100,000 CFU.Each possibility represents a separate embodiment. In other embodiments,the initial concentration of encapsulated microorganisms is about 1 toabout 1×10⁴ CFU, including each value within the specified range, forexample about 1, about 10, about 20, about 30, about 40, about 50, about60, about 70, about 80, about 90, about 100, about 150, about 200, about250, about 300, about 350, about 400, about 450, about 500, about 550,about 600, about 650, about 700, about 750, about 800, about 850, about900, about 950, about 1,000, about 1,500, about 2,000, about 2,500,about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000,about 8,500, about 9,000, about 9,500, or about 10,000 CFU. Eachpossibility represents a separate embodiment. In yet other embodiments,the initial concentration of encapsulated microorganisms is about 10 toabout 1,000 CFU, including each value within the specified range forexample about 10, about 20, about 30, about 40, about 50, about 60,about 70, about 80, about 90, about 100, about 150, about 200, about250, about 300, about 350, about 400, about 450, about 500, about 550,about 600, about 650, about 700, about 750, about 800, about 850, about900, about 950, or about 1,000 CFU. Each possibility represents aseparate embodiment.

Upon rehydration, the selective permeability of the outer shell layerresults in a significant increase in the number of encapsulatedmicroorganisms. As used herein, the term “selective permeability” or“selectively permeable” refers to a shell layer which affords fluidinflow upon rehydration while preventing the release of themicroorganisms and/or nutrients from the particle for a predeterminedperiod of time after rehydration during which microincubation andpropagation of the microorganisms occur. In certain embodiments,microincubation and propagation of the microorganisms result in at leasta 2, 3, 4, 5, 6, 7, 8, 9, or even 10-fold increase in the density orcell count of the microorganisms as compared to naked non-encapsulatedmicroorganisms' density or cell count at a predetermined period of timeafter rehydration. In one embodiment, upon rehydration (fluidabsorption), the concentration of the encapsulated microorganisms isincreased by at least 10-fold before the microorganisms are released tothe surrounding environment. In various embodiments, upon rehydration,the concentration of the encapsulated microorganisms is increased by atleast 20-fold before the microorganisms are released to the surroundingenvironment. In certain embodiments, upon rehydration, the concentrationof the encapsulated microorganisms is increased by at least 30-foldbefore the microorganisms are released to the surrounding environment.

Following microincubation and propagation, a partial or completedegradation or erosion of the outer shell layer of the particle occurs,thereby affording the delayed release of the encapsulatedmicroorganisms. As used herein, the term “delayed release” refers to theinitiation of release of the encapsulated microorganisms after a certainlag time following fluid absorption. Typically, the release of theencapsulated microorganisms is effected from about 24 hours to about 7days following rehydration, including each value within the specifiedrange. For example, the release of the encapsulated microorganisms maybe effected about 24 hours, about 48 hours, about 72 hours, about 96hours, about 120 hours, about 144 hours, or about 168 hours (7 days)following rehydration. Each possibility represents a separateembodiment. In certain embodiments, release of the encapsulatedmicroorganisms is effected from about 24 to about 96 hours followingrehydration, including each value within the specified range, forexample, about 24, about 48, about 72, or about 96 hours followingrehydration. Each possibility represents a separate embodiment. In someembodiments, the concentration of the encapsulated microorganisms isincreased by at least 10-fold 24 hours following fluid absorption. Inother embodiments, the concentration of the encapsulated microorganismsis increased by at least 10-fold 48 hours following fluid absorption. Inyet other embodiments, the concentration of the encapsulatedmicroorganisms is increased by at least 10-fold 72 hours following fluidabsorption. In further embodiments, the concentration of theencapsulated microorganisms is increased by at least 10-fold 96 hoursfollowing fluid absorption. In some embodiments, the concentration ofthe encapsulated microorganisms is increased by at least 20-fold 24hours following fluid absorption. In other embodiments, theconcentration of the encapsulated microorganisms is increased by atleast 20-fold 48 hours following fluid absorption. In yet otherembodiments, the concentration of the encapsulated microorganisms isincreased by at least 20-fold 72 hours following fluid absorption. Infurther embodiments, the concentration of the encapsulatedmicroorganisms is increased by at least 20-fold 96 hours following fluidabsorption. In some embodiments, the concentration of the encapsulatedmicroorganisms is increased by at least 30-fold 24 hours following fluidabsorption. In other embodiments, the concentration of the encapsulatedmicroorganisms is increased by at least 30-fold 48 hours following fluidabsorption. In yet other embodiments, the concentration of theencapsulated microorganisms is increased by at least 30-fold 72 hoursfollowing fluid absorption. In further embodiments, the concentration ofthe encapsulated microorganisms is increased by at least 30-fold 96hours following fluid absorption.

According to certain aspects and embodiments, the outer shell layer ofthe particle is formed from a polymer or polymer precursor configured toundergo a phase transition in response to a stimulus. The stimulus maybe chemically induced or physically induced with each possibilityrepresenting a separate embodiment. Non-limiting examples of stimulusinclude a change in at least one of temperature, pH, light, and electricfield, with each possibility representing a separate embodiment. Thephase transition may be reversible or irreversible with each possibilityrepresenting a separate embodiment. An exemplary phase transitioncomprises the polymerization of a polymer precursor. Another exemplaryphase transition comprises the cross-linking of polymer chains.

Several approaches can be used to provide the particle as describedherein. In one approach the particle can be fabricated fromfluid-degradable material made of single polymers or a plurality ofpolymers at a desired mixing ratio, density and thickness to achieve therequired properties such as permeability and erosion or degradationrate. Synthetic, semi-synthetic or natural polymers can be optionallyblended with proteins for the purpose of improving the process and themechanical properties of fibers forming the outer shell. Polymersutilized in this way include, but are not limited to, polyethylene oxide(PEO), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethyleneglycol (PEG), polycaprolacetone (PCL), polyurethane (PU), polyglycolicacid (PGA), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA),poly-L-lactic acid (PLLA), cellulose derivatives, chitosan, chitin, andhyaluronan (HA), gelatin, alginate polymer, and a mixture or combinationthereof. Each possibility represents a separate embodiment. In oneembodiment, the polymer is other than carboxymethyl cellulose (CMC). Theparticle can be prepared e.g. using extrusion designed to afford anouter shell layer with selective permeability.

In certain embodiments, the outer shell layer comprises polyvinylalcohol (PVA). PVA is known to be available at various degrees ofpolymerization (molecular weight) and/or hydrolysis. Within the scope ofthe present invention is a single grade PVA or a combination of PVAs ofdifferent grades. Each possibility represents a separate embodiment.Grades of PVA suitable as an outer shell layer according to embodimentsof the present invention include, but are not limited to, hydrolyzed PVAhaving a degree of hydrolysis of at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100% (fullyhydrolyzed). Each possibility represents a separate embodiment. Inspecific embodiments, the outer shell layer comprises hydrolyzed PVAhaving a degree of hydrolysis of about 87% to about 99%, including eachvalue within the specified range. Within the scope of the presentinvention is PVA of different chain lengths including, but not limitedto, PVA having a molecular weight ranging from about 1 to about 500kilodaltons (KD), for example about 10 to about 250 KD, preferably about25 to about 125 KD, including each value within the specified ranges.For example, the PVA may have a molecular weight of about 1, about 5,about 10, about 15, about 20, about 25, about 30, about 35, about 40,about 45, about 50, about 55, about 60, about 65, about 70, about 75,about 80, about 85, about 90, about 95, about 100, about 110, about 120,about 130, about 140, about 150, about 160, about 170, about 180, about190, about 200, about 210, about 220, about 230, about 240, about 250,about 260, about 270, about 280, about 290, about 300, about 310, about320, about 330, about 340, about 350, about 360, about 370, about 380,about 390, about 400, about 410, about 420, about 430, about 440, about450, about 460, about 470, about 480, about 490, or about 500 kD. Eachpossibility represents a separate embodiment. In other embodiments, theouter shell layer comprises gelatin. The amount of polymer used forfabricating the particle can vary as is known in the art. Typically theamount of polymer in the outer shell layer ranges from about 0.01% toabout 30% (w/v), for example about 0.1% to about 20% (w/v), or about 1%to about 20% (w/v), including each value within the specified ranges.

Alternatively or additionally, electrical current (electrospinning) canbe used while performing extrusion on synthetic and natural biopolymers,such as nylon 6 and polyethylene oxide (PEO), polyacrylonitrile (PAN)and a composite of polylactide/polyhydroxybutyrate (PLA/PHB) (Wen et al.J. Agric. Food Chem. 65(42), 9161-9179, doi: 10.1021/acs.jafc.7b02956(2017); Mortimer et al., J. Microb. Biochem. Technol. 8, 3; DOI:10.4172/1948-5948.1000285 (2016)).

According to certain aspects and embodiments, the outer shell layer maybe composed of fibrous polymer material, optionally comprising pores ofdifferent sizes. In accordance with these embodiments, the pores may beable to physically trap the encapsulated microorganisms and/or nutrientsfrom being released to the environment while allowing sufficient fluidinflow during rehydration. In some embodiments, the average diameter ofthe pores ranges from about 1 micron to about 100 microns, includingeach value within the specified range, for example about 1, about 5,about 10, about 15, about 20, about 25, about 30, about 35, about 40,about 45, about 50, about 55, about 60, about 65, about 70, about 75,about 80, about 85, about 90, about 95, or about 100 microns. Eachpossibility represents a separate embodiment. In other embodiments, theaverage diameter of the pores ranges from about 20 microns to about 999microns, including each value within the specified range.

According to some aspects and embodiments, the degradation or erosion ofthe outer shell layer may be triggered by changes in pH, light,temperature etc. Each possibility represents a separate embodiment. Inadditional embodiments, the degradation or erosion of the outer shelllayer may be triggered by changes initiated by the propagation of themicroorganisms thereby releasing the microorganisms in a controlledmanner.

In various aspects and embodiments, the composition may further comprisea sustained release agent within the inner core thereby providingprolonged release of the microorganisms after a predetermined lag time.In accordance with these embodiments, following degradation of the outershell layer, the composition serves as a reservoir of microorganismswhich may be slowly released to the target environment. The sustainedrelease agent typically comprises a polymer. Although the inner core andouter shell layer may comprise different polymers, embodiments in whichthe same polymer is used are also contemplated. It is to be understoodthat when using the same polymer in the inner core and outer shelllayer, the physical properties of the inner core and outer shell layermay be distinct such that the outer shell layer is designed to degradeat a different rate than the inner core upon fluid absorption. Two ormore fluid-degradable materials fabricating an inner core and outershell layer having different degradation rates as described herein arecontemplated by the scope of the present invention.

According to further embodiments of the present invention, the outershell layer may be overcoated with at least one other coating layer or aplurality of coating layers, each having a different functionality suchas, but not limited to, different permeability, different sensitivity topH, light, temperature etc. In some embodiments, the layer overcoatingthe outer shell layer may be an adhesive coating layer thereby enablingthe adhesion of the particle to a plant tissue (e.g. seeds).

According to certain aspects and embodiments, there is provided a methodof preparing a dehydrated composition comprising a particleencapsulating one or more microorganisms for delivery of a plurality ofmicroorganisms to a target environment comprising:

-   -   (i) suspending one or more microorganisms in a solution        optionally comprising one or more nutrients;    -   (ii) encapsulating the one or more microorganisms and optional        nutrients in a polymer thereby obtaining a particle composed of        an inner core comprising said one or more microorganisms and        optional nutrients surrounded by an outer shell layer; and    -   (iii) dehydrating the particle.

In some embodiments, step (ii) comprises adding a polymer or polymerprecursor to the suspension comprising the one or more microorganismsand optional nutrients and inducing phase transition to the polymer orpolymer precursor thereby obtaining a particle as described herein. Thephase transition may include polymerization of the polymer precursor orcross-linking of the polymer. Each possibility represents a separateembodiment. The phase transition may be induced physically (e.g. viaheating, or light, or electrical field), or chemically (e.g. addition ofa cross-linking agent). Typically, the phase transition is induced bysubjecting the polymer or polymer precursor to a stimulus, such as, butnot limited to, a change in at least one of temperature, pH, light, andelectric field. Each possibility represents a separate embodiment. Inother embodiments, step (ii) comprises adding (e.g. injecting) thesuspension comprising the one or more microorganisms and optionalnutrients to a preformed particle.

According to embodiments of the present invention, there is provided amethod of delivering a plurality of microorganisms to a targetenvironment. Following application of the dehydrated composition to atarget environment, the composition is hydrated with a suitable amountof fluid (e.g. water) thereby allowing the propagation and subsequentrelease of a plurality of microorganisms from the particle to the targetenvironment in a controlled manner. Target environments include, but arenot limited to, soil, phyllosphere, rhizosphere, sewage reclamation,toxic spill, fermentation, and the gastrointestinal tract. Eachpossibility represents a separate embodiment. A currently preferredtarget environment is a soil environment. In accordance with theseembodiments, the composition comprising a plurality of particles can beused as is by spraying (e.g. foliar spraying)/dusting/pouring to theenvironment (e.g. soil and/or plants) or it can be used in the vicinity(e.g. as a coating, co-seeding etc.) of plant tissue such as seeds.

According to certain aspects and embodiments, seeds (e.g. of commoncrops such as soy, corn, wheat or canola) can be coated with thecomposition of the invention using seed dusting. In other embodiments,gluing the particles to seeds using an adhesive chemical can be done.Following coating, the seeds can be stored until use. Planting andwatering the seeds will rehydrate the particles and lead to thepropagation and release of the microorganisms as is describedhereinabove.

When used to spray/dust crops (e.g. fruit, leaves etc.), the particlescan include a coating for facilitating adhesion of the particles to thecrop tissue (especially when the crop is watered). Such a coating caninclude, for example, carboxymethyl cellulose (CMC) as adhesive materialsuitable for spraying crops. Additionally or alternatively, particlescan be electrically charged to adhere to crops electrostatically whendusting. In certain embodiments, particles can be shaped so as tofacilitate adherence to plant tissue. For example, the outer surface ofthe particle can include protrusions (e.g., stalks, pyramids) that caninteract with plant leaf trichomes to facilitate adherence. A shape thatincreases the surface area can also be used in order to increaseelectrostatic or other interactions with plant tissue.

Referring now to the drawings, FIG. 1 illustrates one embodiment of thepresent invention showing a composition containing one or moremicroorganisms (microbial cells) and nutrients (supplemental materials)within a lumen of a particle (‘dehydrated microfermentor’). Followinghydration (‘water intake’), the nutrients form a medium suitable forpropagation of the one or more microorganisms which then propagate(‘in-field fermentation’) within the particle until reaching a desiredcell count/density before diffusing out to the target environment(‘release’). The release is typically accompanied by outer shell layerdegradation.

Preparation of various compositions of the present invention isdescribed in detail in the Examples section that follows. Briefly, cellsof the microorganism are suspended in solution, optionally furthercomprising nutrients that support propagation. The solution and cellsare encapsulated within a particle (that is prefabricated or fabricatedwithin the solution and cells) and the resulting composition isdehydrated.

FIG. 2 illustrates exemplary main steps of preparing particles withinthe scope of the present invention. The microorganism(s) is grown in afermenter, then harvested and resuspended in a solution or suspensioncontaining a polymer or a polymer precursor, as well as optionalnutrients to support cell growth following rehydration. Polymerizationor cross-linking is then induced physically and/or chemically as isknown in the art, for example, using spinning and/or modified conditionssuch as pH, temperature, and/or the addition of a cross-linking agent(see Examples section), thereby encapsulating the microorganism(s) andoptional nutrients. The suspension containing the encapsulatedmicroorganism(s) and optional nutrients is then dehydrated to powderform, optionally followed by additional coating or packaging for storageand transport to a target environment. Upon application to a targetenvironment, wetting (rehydration) and reconstitution is then effectedto induce microfermentation, degradation and controlled release asillustrated in FIG. 1.

As used herein and in the appended claims, the term “about” refers to±10%.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural references unless the context clearly dictatesotherwise. Thus, for example, reference to “a layer” includes aplurality of such layers and equivalents thereof known to those skilledin the art, and so forth. It should be noted that the term “and” or theterm “or” are generally employed in its sense including “and/or” unlessthe context clearly dictates otherwise.

It will be apparent to one of ordinary skill in the relevant art thatsuitable modifications and adaptations to the compositions, methods,processes, and applications described herein can be made withoutdeparting from the scope of any embodiments or aspects thereof. Thecompositions and methods provided are exemplary and are not intended tolimit the scope of any of the specified embodiments. All of the variousembodiments, aspects, and options disclosed herein can be combined inany and all variations or iterations. The scope of the compositions,methods, and processes described herein include all actual or potentialcombinations of embodiments, aspects, options, examples, and preferencesherein described. The exemplary compositions described herein may omitany component, substitute any component disclosed herein, or include anycomponent disclosed elsewhere herein. The ratios of the mass of anycomponent of any of the compositions disclosed herein to the mass of anyother component in the composition or to the total mass of the othercomponents in the composition are hereby disclosed as if they wereexpressly disclosed. Furthermore, the foregoing discussion discloses anddescribes merely exemplary embodiments. Additional objects, advantages,and novel features of the present invention will become apparent to oneordinarily skilled in the art upon examination of the followingexamples, which are not intended to be limiting.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non-limiting fashion.

Example 1: Fermentation of Bacterial Strains Prior to Encapsulation

One strain (Pseudomonas chlororaphis, Phylum Proteobacteria) was testedfor microfermentation in soil. It is to be understood that other strainse.g. Arthrobacter globiformis (Phylum Actinobacteria) and Bacillusendophyticus (Phylum Firmicutes) can also be used. Strains are removedfrom cryogenic or lyophilized storage. Inocula are prepared by eitherdirect transfer to a suitable liquid media (LB, NB, TSB, R2A or otherbacteriological growth medium) and incubation in a shaker incubator for10-15 hours at 200 rpm and 28° C., or transfer to an agar plate media(LB, NB, TSB, R2A or other bacteriological growth medium) and growth for24-48 hours before selecting colonies for transfer to a suitable liquidmedia. The inocula are transferred to a shake flask or a fermenter.Fermenter includes standard pH and dissolved oxygen (DO) probes;temperature and pH control; and programmed nutrient feeding. Mediacontaining a suitable carbon source such as glucose, sucrose, molasses,cellulose or starch; a nitrogen source such as peptones, or peptides;additional nutrients required by the organism such as minerals andvitamins; and antifoaming compounds, such as silicones or polypropyleneglycol P2000, are sterilized in the fermenter or in an autoclave, andtransferred aseptically to the sterile flask or fermenter to initiategrowth. Before harvest after 12-48 hours, the flask or fermenter issampled and evaluated for cell density. Growth is continued in the flaskor fermenter until optical density (OD) at 600 nm is greater than 10,indicating that the colony forming units (CFUs) are greater than 1×10⁹CFUs/ml. The flask or fermenter contents are centrifuged at 10,000 g and4° C. for 10 minutes, to recover the bacteria. From a fermenter, abacterial paste of more than 30 g/liter is recovered. The sample isdiluted 1:10 in phosphate buffered saline (PBS, pH 7.0) or other desiredsolution. A small sample (1 ml) is serially diluted 10-fold severaltimes in sterile PBS. The resulting diluted bacterial suspensions arethen plated on sterile LB, NB, TSB, R2A or other bacteriologicalculturing medium. After 48 hours of incubation at 28° C. in the dark,colony forming units (CFUs) in each dilution are enumerated. Calculationof CFUs per 1 ml is performed by multiplying CFUs in each dilution bythe dilution factors and averaging the results obtained from thedifferent dilutions.

Static fermentation can also be used, where the inocula is spread onagar media in bottles or plates. Growth is continued until obtainingrelevant physiological stage, and the microorganism is collected by agarwash with phosphate buffered saline (PBS, pH 7.0) or other desiredsolution. A small sample (1 ml) is serially diluted 10-fold severaltimes in sterile PBS for CFU assay as described above.

Example 2: Formulation with Chemical Additives without Encapsulation

Three methods are employable as follows:

Method 1: Bacteria are harvested by centrifugation at 10,000 g and 4° C.for 10 min. The pellet is mixed with 20% Microcel-E or Sipernat 22S, 10%mannitol, maltodextrin, sorbitol, and/or skim milk powder using a coffeegrinder. The wet powder is dried in a force air oven at 40° C.Method 2: Bacteria are harvested and re-suspended in water to a tenth ofthe initial (fermentation) volume. The bacterial suspension is mixedwith 5% mannitol, maltodextrin, sorbitol, and/or skim milk powder and issprayed onto Microcel-E or Sipernat 22S using a Strea-1 fluid bed dryer.Inlet temperature is kept at 40° C.Method 3: Bacteria are harvested and re-suspended in water to a tenth ofthe initial (fermentation) volume. The bacterial suspension is mixedwith 5% mannitol, maltodextrin, sorbitol, skim milk powder, and/or gumarabic or 3% skim milk powder and 2% gum arabic and is dried with aBuchi B-290 spray dryer. Inlet temperature is kept at 60° C.

Example 3: Encapsulation Electrospinning

Generation of electrospun capsules is conducted in a manner known in theart, such as in Damasceno et al. (Can. J. Microbiol. 59, 716-719(2013)). Bacteria are harvested by centrifugation at 10,000 g and 4° C.for 10 min and the paste is re-suspended in sterile 0.9% NaCl,containing glucose, sucrose, molasses, cellulose or starch, at aconcentration of up to 100 μg/liter, and an extract or digest of casein,soy, corn and/or yeast at a concentration of 20-200 μg/liter. Asterilized solution of 30% PVA in distilled water is added to thesuspended cells to a final concentration of 15% PVA. To polymerize thecapsules around the microbial cells, the solution is transferred to 5 mlsyringes, and extruded at a syringe descent rate of 0.01 mm/s through aneedle at a voltage of 22 kV and into a collection tube at a distance of14 cm, in a manner calculated to create capsules of 50 μm diameter, witha permeability of 0.5 kDa molecular weight cut-off (MWCO). The capsulesare dried in the collection tubes for 2 hours.

Alginate Encapsulation

Method 1: Bacteria are harvested by centrifugation at 10,000 g and 4° C.for 10 min and the paste is re-suspended in sterile 0.9% NaCl. 5% Skimmilk powder, glucose, maltodextrin, NB and/or LB are added to thebacterial suspension prior to the mixing with the alginate solution. Thesuspension is adjusted to a cell concentration of ˜1×10⁸ CFU/ml. A 2%(w/v) low viscosity alginate solution is prepared in dH₂O and mixed 1:10with the bacterial suspension. The suspension is then extruded throughan 80 micron nozzle of a Buchi B-390 Encapsulator (Buchi GmbH, Flawil,Switzerland) into a beaker filled with 0.2 M CaCl₂ under fast agitation.Beads are hardened in CaCl₂ for 30 min, filtered off with a vacuum pump,rinsed with dH₂O, filtered again and dried by lyophilization.

Method 2: Bacteria are harvested by centrifugation at 10,000 g and 4° C.for 10 min and the paste is re-suspended in sterile 0.9% NaCl. Thesuspension is adjusted to a cell concentration of ˜1×10⁸ CFU/ml. 5% Skimmilk powder or glucose, maltodextrin, NB and/or LB are added to thebacterial suspension prior to the mixing with the alginate solution. A2% (w/v) low viscosity alginate solution is prepared in dH₂O and mixed1:10 with the bacterial suspension. The mixture is added dropwise withthe aid of a 10-ml sterile syringe into 0.2 M CaCl₂ that is gentlystirred at room temperature. The beads are maintained in the solution atroom temperature for an additional 1 hour to obtain regular solid beads.The CaCl₂ solution is then pumped out, and the beads are washed twicewith tap water. The beads are strained and dried in a forced air oven at30° C.

Drop Encapsulation

A solution of 20% PVA of different grades (W/V; polymer/DDW), and/orgelatin was prepared according to manufacturer's recommendations.Several modifications, such as use of growth media including NB or LB,were optionally made. The suspension was then sterilized. Bacterialsuspension was prepared by re-suspension of bacteria in relevant bufferor growth media. The suspension was adjusted to cell concentrations of1×10²-1×10¹⁰ CFU/ml. Drops of polymer suspension were plated on glass.After a few minutes of polymerization, the bacterial suspension wasinjected into the polymers in each drop. The drop capsules were dried ina forced air oven at 30° C. for 3-4 hours. The flat capsules containingbacteria were removed by knife from the glass for immediate use orstorage.

Example 4: Cell Viability Assessment in Storage

Formulation powder (100 mg) is suspended in 1 ml phosphate bufferedsaline (pH 7.0; PBS) and is 10-fold serially diluted in sterile PBS. Theresulting diluted bacterial suspensions are then plated on sterile LB,NB, TSB, R2A or other bacteriological culturing medium. After 48 hoursof incubation at 28° C. in the dark (thereby allowing degradation of thecapsules), colony forming units (CFUs) in each dilution are enumerated.Calculation of CFUs per 1 gram of formulation is performed bymultiplying CFUs in each dilution by the dilution factors and averagingthe results obtained from the different dilutions of each formulationpowder.

Example 5: Cell Growth Assessment in Soil/on Seed

Formulation powder (100 mg) is mixed with 1 gram of soil in a sealed 15ml tube. Then, 500 μl of tap water are added into the mixture and thetube is incubated for 5 days at 17° C. in the dark. Strain viability isassessed every 24 hours for 4 days. At each time point, sterile PBS isadded to a tube to a final volume of 5 ml, the tube is vigorously mixedfor 10 min and its contents are 10-folds serially diluted several timesin sterile PBS. The resulting diluted bacterial suspensions are platedon sterile LB, NB, TSB, R2A or other bacteriological culturing medium.After 48 hours of incubation at 28° C. in the dark, colony forming units(CFUs) in each dilution are enumerated. Calculation of CFUs per 1 gramof formulation is performed by multiplying CFUs in each dilution by thedilution factors and averaging the results obtained from the differentdilutions of each tube.

The encapsulation of the present invention is envisioned to affordincrease in growth and viability in soil relative to nakednon-encapsulated microorganisms as well as microorganisms encapsulatedwith hitherto known packaging methods.

Example 6: Cell Growth at Low and High Initial Concentrations in Soil

Pseudomonas chlororaphis bacteria (2 mg) encapsulated using the dropencapsulation method described hereinabove in Example 3 were mixed in 2grams of soil in a sealed 50 ml tube. Then, 900 μl of tap water wereadded into the mixture and the tube was incubated for up to 6 days at19° C. in the dark. Bacterial viability was assessed every 24 hours over4 days. At each time point, sterile PBS was added to a tube to a finalvolume of 10 ml, the tube was vortexed for 30 s and its contents wereserially diluted 10-fold several times in sterile PBS. The resultingdiluted bacterial suspensions were plated on sterile R2A or otherbacteriological culturing medium containing selective antibiotics. After48 hours of incubation at 28° C. in the dark, colony forming units(CFUs) in each dilution were enumerated. Calculation of CFUs per 1 mg ofencapsulated particle was performed by multiplying CFUs in each dilutionby the dilution factors and averaging the results obtained from thedifferent dilutions of each tube.

The initial concentration of the bacteria was assayed by taking 1 mg ofencapsulated particles per 1 ml sterile PBS, incubated for 60 min at RT,then serially diluted and plated on sterile R2A or other bacteriologicalculturing medium containing selective antibiotics. After 48 hours ofincubation at 28° C. in the dark, CFUs in each dilution were enumerated.

FIGS. 3A-3B show graphs which present comparative results of propagationin soil of naked (▪) or encapsulated Pseudomonas chlororaphis indifferent polymers (♦ PVA 87 h Mw=85K-124K; ● 1:1 PVA 87 h Mw=85K-124Kand PVA 99 h Mw=31K-50K; ▴ gelatin; ×1:1 PVA 99 h Mw=31K-50K andgelatin; and

1:1:2 PVA 87 h Mw=85K-124K, gelatin, and PVA 99 h Mw=31K-50K). FIG. 3Ashows the propagation at initial concentrations of 10²-10⁵ CFU per 2grams of soil and FIG. 3B shows the propagation at initialconcentrations of 10⁵-10⁶ CFU per 2 grams of soil. Whereas at initialconcentrations of 10⁵-10⁶ CFU per 2 grams of soil both naked andencapsulated bacteria showed propagation, at initial concentrations of10²-10⁵ CFU per 2 grams of soil only the encapsulated bacteria showedpropagation while the naked bacteria lost viability. These resultsdemonstrate the advantage conferred by the encapsulation of the presentinvention at low initial concentrations.

FIG. 4 shows the comparative results of propagation in soil ofPseudomonas chlororaphis at initial concentrations of 10³-10⁴ CFU per 2grams of soil encapsulated in PVA 99 h Mw=31K-50K (▴) and in CMC (▪)over 144 hours. Whereas the encapsulation in PVA showed bacterialpropagation, the encapsulation in CMC yielded loss in viability, showingthat not all types of encapsulation can provide bacterial propagation inthe relevant environment. Without being bound by any theory or mechanismof action, it is contemplated that the CMC encapsulation did not affordproper propagation due to its higher solubility in water therebyresulting in the premature degradation of the capsule.

Example 7: Bacterial Growth within a Polymer Capsule

Pseudomonas chlororaphis bacteria cells were encapsulated within thepolymer using the drop encapsulation method as described hereinabove inExample 3. The dried capsules were then placed in the middle of R2Aplates or any other nutrient plate and incubated at 28° C. for 24-72hours. At time 0 and every 24 hours thereafter, 100 μl of DDW werepipetted on the edge of the plate, to provide water at diffusion rate.After 48 hours, the edges of the capsule were no longer smooth,suggesting the release of the bacteria.

FIGS. 5A-5C illustrate the growth of Pseudomonas chlororaphis within thecapsule, at time 0 (T_(0 h); FIG. 5A), after 24 hours (T_(24 h); FIG.5B), and after 48 hours (T_(48 h); FIG. 5C). The light areas are thecapsules, which expand with hydration. The rougher edges of the lightarea at 48 hours represent bacterial growth. Identity of growingbacteria was demonstrated by the development of color. Specifically,Pseudomonas chlororaphis at high bacterial dose develop a yellow toorange pigment color. At the indicated time points, the plates werephotographed at high resolution in order to quantify the intensity ofthe developed color in the light areas. The color was quantified usingthe IMAGEJ software, as the intensity within the sampled area in thecolor hue range of 25-50 (representing yellow-orange pigmentation ofPseudomonas chlororaphis) multiplied by the percentage of colored areawithin the sampled area, and is shown on the bottom left corner of eachfigure. The intensity of the color at time 0 (T_(0 h)), after 24 hours(T_(24 h)), and after 48 hours (T_(48 h)) was 556.1, 922.1, and 3275.9,respectively.

Example 8: Accelerated Model of Bacterial Release from a Polymer

In order to demonstrate release of the bacteria from the capsule, anaccelerated model was used. The accelerated model affords the releasefrom the capsule starting immediately after hydration and up to 70minutes thereafter. It is contemplated that the release in soil is muchslower, thereby enabling the bacteria to propagate within the capsuleprior to release.

The accelerated model was prepared as follows: a modification of thedrop encapsulation method described hereinabove in Example 3 was appliedsuch that instead of preparing the drops on glass, the drops wereprepared on the well corners of a 24 well plate and remained on theplate without removal. The bacteria used for this assay were GFPlabeled. At time zero, 900 μl of DDW were added to each well and GFPemission was measured at 509 nm after excitation at 395 nm. Reads wererecorded by 3 successive flashes at intervals of 5 min. Each data pointrepresents an average of 4 independent repeats, with 3 flashes perrepeat per time point.

FIG. 6 shows graphs of bacterial release from various capsules (▴ PVA31-50K, 99 h capsule; ♦ gelatin capsule; and ▪ 1:1 PVA 85-124K, 87 h andPVA 85-124K, 99 h capsule) in the accelerated model. The results showthe effective release of the bacteria from all capsules followingrehydration. Naked bacteria used as control (●) showed constant GFPintensity.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

1-32. (canceled)
 33. A dehydrated composition comprising a particleencapsulating one or more microorganisms, wherein the particle iscomposed of an inner core comprising the one or more microorganismssurrounded by an outer shell layer, wherein said outer shell layer isselectively permeable to a rehydrating fluid, and wherein upon fluidabsorption, said outer shell layer degrades at a predetermined ratethereby releasing a plurality of microorganisms to the surroundingenvironment in a controlled manner after a predetermined lag time,wherein the encapsulated microorganisms are present in the dehydratedcomposition at an initial concentration of less than 1×10³ colonyforming units (CFU) per particle, and wherein following fluidabsorption, the concentration of the encapsulated microorganisms isincreased by at least 10-fold before the microorganisms are released tothe surrounding environment.
 34. The composition of claim 33, whereinthe outer shell layer comprises a naturally occurring polymer, asynthetic polymer, or a semi-synthetic polymer; or wherein the outershell layer comprises a water-degradable polymer; or wherein the outershell layer comprises a polymer selected from the group consisting ofpolyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene oxide(PEO), polyethylene glycol (PEG), polycaprolactone (PCL), polyurethane(PU), polyglycolic acid (PGA), polylactic acid (PLA),polylactic-co-glycolic acid (PLGA), poly-L-lactic acid (PLLA), cellulosederivatives, chitosan, chitin, hyaluronan (HA), gelatin, nylon 6,polyacrylonitrile (PAN), polylactide/polyhydroxybutyrate (PLA/PHB),alginate polymer, and a mixture or combination thereof.
 35. Thecomposition of claim 34, wherein the polymer is polyvinyl alcohol (PVA)or gelatin.
 36. The composition of claim 33, wherein the outer shelllayer is designed to be digested or consumed by the encapsulatedmicroorganisms; or wherein the outer shell layer is formed from apolymer or polymer precursor configured to undergo a phase transition inresponse to a stimulus, wherein the stimulus comprises a change in atleast one of temperature, pH, light, and electric field.
 37. Thecomposition of claim 33, wherein the particle is less than 100 micronsin diameter.
 38. The composition of claim 33, wherein the release of themicroorganisms initiates about 24 to about 96 hours following fluidabsorption; and/or wherein the concentration of the encapsulatedmicroorganisms is increased by at least 10-fold about 24 hours followingfluid absorption.
 39. The composition of claim 33, wherein the innercore further comprises one or more nutrients comprising at least one ofa carbon source, a nitrogen source, and a phosphorous source.
 40. Thecomposition of claim 33, wherein the microorganisms comprise endophyticbacteria.
 41. The composition of claim 33 further comprising an adhesivecoating layer over the outer shell layer.
 42. The composition of claim33, wherein the inner core further comprises a fluid-degradable materialhaving a different degradation rate than the outer shell layer uponfluid absorption.
 43. The composition of claim 33, comprising aplurality of particles.
 44. The composition of claim 43, wherein theplurality of particles is further surrounded by an additional coatinglayer.
 45. A plant tissue coated with or in the vicinity of thecomposition of claim
 33. 46. The plant tissue of claim 45, being a plantseed.
 47. A method of preparing a dehydrated composition comprising aparticle encapsulating one or more microorganisms as claimed in claim33, the method comprising: (i) suspending one or more microorganisms ina solution optionally comprising one or more nutrients; (ii)encapsulating the one or more microorganisms and optional nutrients in apolymer, thereby obtaining a particle composed of an inner corecomprising said one or more microorganisms and optional nutrientssurrounded by an outer shell layer; and (iii) dehydrating the particle.48. The method of claim 47, wherein step (ii) comprises adding a polymeror polymer precursor to the suspension comprising the one or moremicroorganisms and optional nutrients of step (i) and inducing phasetransition to the polymer or polymer precursor, thereby obtaining aparticle composed of an inner core comprising said one or moremicroorganisms and optional nutrients surrounded by an outer shelllayer.
 49. The method of claim 48, wherein the step of inducing phasetransition to the polymer or polymer precursor comprises polymerizationof the polymer precursor or cross-linking of the polymer; or wherein thestep of inducing phase transition to the polymer or polymer precursorcomprises subjecting the polymer or polymer precursor to a stimulus,wherein the stimulus comprises a change in at least one of temperature,pH, light, and electric field.
 50. The method of claim 47, wherein step(iii) is performed to a water content of about 0.5% to about 10% byweight of the total dehydrated composition.
 51. A method of delivering aplurality of microorganisms to a target environment, comprisingrehydrating a dehydrated composition as claimed in claim 33 with asuitable amount of fluid, thereby allowing the propagation andsubsequent release of a plurality of microorganisms from the particle toa target environment in a controlled manner.
 52. The method of claim 51,wherein the target environment comprises soil, phyllosphere,rhizosphere, sewage reclamation, toxic spill, fermentation, or thegastrointestinal tract.